Objective Eel is a major freshwater aquaculture economic species in China. Eel is rich in nutrients and delicious meat, which is very popular among consumers. However, with the continuous development of intensive aquaculture mode, there are more and more kinds of diseases in eel culture, among which the danger of viral diseases is particularly serious. Anguillid herpesvirus (AngHV) poses a significant threat to eel (Anguilla) breeding, often resulting in economic losses. Developing rapid detection technologies for AngHV is crucial for disease prevention and control in China’s eel industry.

Methods AngHV DNA was extracted from liver and gill tissues of diseased eels in the laboratory using a DNA extraction kit. This study designed amplification primers and probes targeting AngHV ORF55 to establish basic RPA and RPA coupled with lateral flow detection (RPA-LFD) methods. The effects of time and temperature variations on RPA and RPA-LFD amplification were investigated. The sensitivity, specificity, and clinical applicability of both methods were compared.

Results Both basic RPA and RPA-LFD assays amplified products of 394 bp within 20 to 40 minutes.The basic RPA assay time exceeded 80 min, which easily led to the appearance of stray bands. When the RPA-LFD detection time was extended to 160 min, the negative control group was prone to false positives. Temperature variations between 37 °C to 43 °C minimally affected amplification efficiency. It indicated that the change in temperature has little effect on the amplification results. Specificity tests showed no cross-reactivity with Eel circovirus, Cyprinid herpesvirus, and Koi herpesvirus. RPA-LFD exhibited high sensitivity with a detection limit of 1.32×10⁰ copies/mL, while basic RPA had a limit of 1.32×10³ copies/mL. Testing 20 clinical eel samples yielded consistent results between the two assays, with a 30% positive detection rate, indicating promising clinical applicability for both methods.

Conclusion In this study, the primers of basic RPA and RPA-LFD were designed in the conserved region of AngHV genes, and the reaction time and temperature were optimised, and finally the rapid detection methods of basic RPA and RPA-LFD for AngHV were established. Both methods have the advantages of rapidity, sensitivity, specificity and good clinical application. The basic RPA and RPA-LFD assays for AngHV detection are suitable for use in grassroots research institutes, eel product processing farms, and breeding facilities. These methods expand the toolkit for detecting eel herpesvirus, enhancing disease management in the eel industry.